CN102725411B - OsMPT gene modifying plant architecture (plant shape) and increasing yield, and use thereof - Google Patents
OsMPT gene modifying plant architecture (plant shape) and increasing yield, and use thereof Download PDFInfo
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- 239000004009 herbicide Substances 0.000 description 1
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- 230000002209 hydrophobic effect Effects 0.000 description 1
- YQYJSBFKSSDGFO-FWAVGLHBSA-N hygromycin A Chemical compound O[C@H]1[C@H](O)[C@H](C(=O)C)O[C@@H]1Oc1ccc(\C=C(/C)C(=O)N[C@@H]2[C@@H]([C@H]3OCO[C@H]3[C@@H](O)[C@@H]2O)O)cc1O YQYJSBFKSSDGFO-FWAVGLHBSA-N 0.000 description 1
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- 229930027917 kanamycin Natural products 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
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- 238000005259 measurement Methods 0.000 description 1
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- 230000000050 nutritive effect Effects 0.000 description 1
- 229940099990 ogen Drugs 0.000 description 1
- 238000007500 overflow downdraw method Methods 0.000 description 1
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- 229940049547 paraxin Drugs 0.000 description 1
- 210000004332 phalangeal cell Anatomy 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 238000005554 pickling Methods 0.000 description 1
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- 230000032361 posttranscriptional gene silencing Effects 0.000 description 1
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- 239000000126 substance Substances 0.000 description 1
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- 229910052717 sulfur Inorganic materials 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 229960004799 tryptophan Drugs 0.000 description 1
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- 239000013603 viral vector Substances 0.000 description 1
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Abstract
The present invention relates to a rice (Oryza sativa)-induced Oryza sativa modifier of plant type (OsMPT) protein which is involved in adjusting tillering angle or angle of attachment between a leaf and a stem, a gene encoding the protein, a recombinant plant expression vector including the gene, a plant transformed by the recombinant plant expression vector, a method for modifying architectures of plants by adjusting the level of the gene in the cells thereof, to a method for manufacturing an architecturally modified plant transformed by the gene, to a plant with increased yield which is manufactured by the method, and to a composition containing the gene for modifying the architectures of plants and increasing the yield thereof.
Description
Technical field
The present invention relates to change plant profile and increase output, derive from OsMPT gene of paddy rice (Oryza sativa) and uses thereof, more specifically, relate to the recombinant plant expression vector that comprises OsMPT gene, transform the plant materials of described recombinant plant expression vector, regulate the interior level (level) of cell of described gene to change plant profile (architectures), and increase the method for output, thereby utilize the method for the plant of described gene-transformed plant preparation structural modification and the plant of increase output, and comprise described gene for changing the profile of plant and increasing the composition of output.
Background technology
Paddy rice (Oryza sativa) is most important food crop, world population more than 1/3 using it as main food source.Because the consumption of paddy rice sharply increases, require paddy rice to there is more output.Therefore, require to have the rice varieties that higher productivity and production has more stability [Khush 1997, and molecular biology of plants 35,25-34(Khush 1997, Plant Mol.Biol.35,25-34)].The structure that is called the plant materials over-ground part of plant profile (architectures) is brought important impact to productivity.Especially, tillering angle is the key property that determines paddy rice profile (architecture), and wider tillering angle increases the shade being caused by leaf, and on the contrary, narrower tillering angle is conducive to high-density planting.Therefore, relatively high efficiency carry out high yield cultivation vertical growth type (narrow tillering angle) and become target, and continued to continue to use by ancients.But the molecule that vertical growth is relevant up to now and Genetic Control mechanism are still not clear.
Although the profile of plant (architectures) is subject to the impact of environmental factors, mechanisms of gene regulation plays important effect to the growth regulating of plant.There are some directly to regulate the relevant gene of rice tillering angle to be disclosed.PROG1 (PROSTRATE GROWTH1) a kind of C2H2 zinc finger protein matter of encoding, prog1 varient becomes vertical growth type owing to having lost prog1 function, becomes the paddy rice with more rice grain numbers and higher output yield.[Tan etc. 2008, natural genetics 40,1360-1364(Tan etc. 2008, Nat.Genet.40,1360-1364)] due to TAC1(TillerAngle Control 1, tillering angle controls 1) transgenation become and there is the intensive plant extremely uprightly tillering [Yu etc. 2007, plant magazine 52,891-898(Yu etc. 2007, Plant J.52,891-898)].LAZY1, TAC1 and PROG1, at leaf sheath pulvinus (leaf sheath pulvinus), express strong in the junction surface of tiller base portion and blade and leaf sheath (laminar joint).
Summary of the invention
The technical problem solving
The present invention proposes in view of above-mentioned requirements, the inventor etc. by identifying OsMP gene mutation body and studying from Ds transposon gene mutant library (pool), preparation OsMPT crosses expression body, understand the effect of OsMPT gene, and the method for utilizing said gene to change plant profile and the method that increases output are provided.
Technical scheme
In order to address the above problem, the invention provides the OsMPT(plant type of rice improvement (Oryza sativa Modifier of Plant Type) that derives from paddy rice (Oryza sativa)) protein.
In addition, the invention provides the gene of coding OsMPT protein.
In addition, the invention provides the recombinant plant expression vector that comprises OsMPT gene.
In addition, the invention provides the plant materials that transforms described recombinant plant expression vector.
In addition, the invention provides the method that changes plant profile by level in adjusting OsMPT polypeptide cell.
In addition, the present invention is by using OsMPT gene-transformed plant, thereby the manufacture method of the plant that profile is changed is provided.
In addition, the invention provides comprise OsMPT gene for changing the composition of plant profile.
In addition, the invention provides and use OsMPT gene-transformed plant, thereby make the preparation method of the plant that output increases.
In addition, the invention provides the plant of the output increase of preparing by method.
In addition, the invention provides the composition for increasing plant biomass that comprises OsMPT gene.
Beneficial effect
In the present invention, the level in OsMPT protein cell that regulates is provided, or with OsMPT gene-transformed plant, change the method for plant profile, by the method, can prepare the heterophyte that tillering angle or leaf are attached to the angular distortion on stem, particularly above-mentioned heterophyte can become the plant of high yield.
Accompanying drawing explanation
Fig. 1 represents the orientations (Alignment) of the aminoacid sequence of OsMPT and Arabidopis thaliana SGR5.Identical amino acid represents by black box, and similarly amino acid represents by grey box.Sequence, Zinc finger domain and coiled coil (coiled coil) territory that is speculated as nuclear location signal represents with underscore.OsMPT has 42% consistence and 54% similarity with SGR5 on amino acid levels.Above-mentioned two kinds of genes have 84% consistence except 22 amino acid between first and second zinc refer to.
Fig. 2 represents that Ds inserts the expression of allelic genome structure and OsMPT::Ds and its recovery mutant (rev.).By Ds being moved to again to original position (Ori.Ds), obtain recovery mutant allele.Original insertion varient and its recovery mutant have been checked to OsMPT expression.
Fig. 3 represents non-transformed contradistinction system (WT: left side), in the middle of OsMPT::Ds() and recovers mutant (Rev.: tillering and the angle of leaf segment (laminar joint, blade and leaf sheath intersection) right side).Tillering angle is measured to outermost tillering from main shaft.Leaf segment angle is measured to the foot of blade from stem.
Fig. 4 represents that OsMPT crosses the erect type plant materials of expression body (OX).Cross expression body by reducing to tiller and the angle of leaf segment, thereby become erect type plant materials.The degree of above-mentioned angle depends on OsMPT mRNA level.The marking hybridization (Northern blot) of bottom is analyzed and is shown that each OsMPT crosses the expression level of expression body (OX).
Fig. 5 represents that BR mutant (d11-1, d61-1) and OsMPT cross expression body (OX).BR mutant and OsMPT cross expression body and show as similar erect type plant materials.D11 catalysis in BR building-up process (6-deoxidation) 3-Standone with CYP724B1 or DWARF4L name) typhasterol of → (6-deoxidation) typhasterol ((6-deoxo) 3-dehydroteasterone) → (6-deoxo)) step.D61-1 is the weak allelotrope of the paddy rice bri1 homology of BR-receptor kinase.
In Fig. 6, contrast the phenotype of the dual gene mutation body between OsMPT and BR signal (d61-1) or synthetic (d11-1) mutant.Ripe plant materials is separated by the hybridization between OsMPT and d61-1 or d11-1.
Fig. 7 has represented to use the erect type plant materials of OsMPT.Plant materials during dense planting with high yield potentiality is crossed in expression system (OX) out selected at OsMPT.The Eastern Jin Dynasty is the mutation of good japonica rice (Japonica) paddy rice.
Fig. 8 represents that the OsMPT in packing crosses expression system (transplanting latter approximately 2 months).(a) and (c) represent successively the Eastern Jin Dynasty paddy rice (WT: non-transformed contradistinction system) cross expression system (OX19) with OsMPT from (30x15cm) plantation with standard species planting distance.(c) red vertical dotted line represents the boundary line between WT and OX19.(b) and (d) Eastern Jin Dynasty paddy rice and the OsMPT of expression dense planting (15x15cm) cross expression system (OX14).(a) and (b) be illustrated in the captured photo in position that likens object height to, (c) and (d) at the captured photo of the similar height location of same crop.
Fig. 9 represents the phenotype evaluation result of conversion of plant.By all data of crossing expression system, divided by non-transformed contradistinction system (WT), be converted to the percentage of prompting in table 1.Line (100%) top represents the value higher than non-transformed contradistinction system (WT), and bottom represents lower than the low value of non-transformed contradistinction system (WT).
Embodiment
In order to reach the object of the invention, provide aminoacid sequence by SEQ ID No.1 to form, derive from the OsMPT(plant type of rice improvement (Oryza sativa Modifier of Plant Type) of paddy rice (Oryasativa)) protein.
According to the scope of OsMPT protein of the present invention, comprise separated having by the protein of aminoacid sequence shown in SEQ ID No.1 and the function coordinator of above-mentioned protein from paddy rice (Oryza sativa).So-called " function coordinator " refers to amino acid whose additional, replacement or solid result, by the aminoacid sequence shown in SEQ ID No.1, at least there is more than 70% sequence identity with above-mentioned, more preferably more than 80%, more preferably more than 90%, more preferably more than 95%, refer to the protein in fact with the protein shown in SEQ ID No.1 with identical physiologically active.
In addition, the invention provides the gene of the described OsMPT protein of coding.Genomic dna and cDNA that gene of the present invention comprises coding OsMPT protein.Preferably, gene of the present invention can comprise by the cDNA base sequence shown in SEQ ID No.2 and by the genomic dna base sequence shown in SEQ ID No.3.In addition, the varient of described base sequence within the scope of the present invention.Particularly, described gene can comprise the base sequence that has respectively more than 70% sequence identity with the base sequence of SEQ ID No.2 or SEQ ID No.3, be preferably more than 80%, and more preferably 90%, most preferably be more than 95%.Polynucleotide " % of sequence identity " are by relatively sequence and the comparison domain of two optimal alignment are determined, in comparison domain, a part for polynucleotide sequence is compared to comprise with the reference sequences (do not comprise and add or delete) of the optimal alignment of two sequences and is added or deletion (that is, breach).
OsMPT gene of the present invention is expressed in special pulvinus (pulvinus) mode.
In addition, the invention provides the recombinant plant expression vector that comprises OsMPT gene of the present invention.In recombinant plant expression vector of the present invention, described OsMPT gene can consist of the base sequence of SEQ ID No.2.
Term " restructuring " refers to cellular replication heterologous nucleic acid, or expresses above-mentioned nucleic acid or express the cell by the protein of peptide, xenogenesis peptide or heterologous nucleic acid coding.Reconstitution cell can be in the form of justice or antisense by not found gene or gene fragment expression in the natural form of above-mentioned cell.In addition, reconstitution cell can be expressed the gene of finding from the cell of native state, but said gene is being reintroduced back in cell by artificial means of distortion.
Term " carrier " is used when the DNA fragmentation to intracellular delivery, nucleic acid molecule.Carrier can repetition DNA, and produces separately in host cell again.Term " carrier " conventionally exchanges and uses with " carrier ".Term " expression vector " refers to and comprises for expressing the recombinant DNA molecules of target code sequence and the necessary suitable nucleotide sequence of the encoding sequence that can be connected at specific host biology with starting.Available promotor in eukaryotic cell, enhanser, termination signal and polyadenylic acid signal are known.
The preferred example of recombinant vectors is can be by the part of himself while being present in suitable host as Agrobacterium tumefaciens, the Ti-plasmid vector that so-called T-region can be shifted to vegetable cell.The Ti-plasmid vector of other type (with reference to No. 0 116 718 B1, EP) utilizes at present in the following areas.Vegetable cell or hybrid DNA are suitably transferred to and can be produced new plant hybrid DNA sequence is transferred in protoplastis in Plant Genome.The most preferred form of Ti-plasmid vector is No. 0 120 516 B1, EP and United States Patent (USP) the 4th, so-called double base (binary) carrier of request in 940, No. 838.In the present invention, preferred binary vector can be pGA1611, but is not limited to this.According to the present invention, can be used for by DNA be incorporated into other preferred carrier in plant host be come from double-stranded plant virus (such as, CaMV) and the virus vector of strand virus, Geminivirus (Gemini virus) etc., for example can from incomplete property plant viral vector, select.The use of such carrier is especially favourable when being difficult to plant host to do suitable conversion.
Preferably, expression vector should comprise more than one selected marker.Above-mentioned mark normally has the nucleotide sequence of the characteristic of available chemical process selection, is equivalent to all genes that transformant can be distinguished from non-transformed cell.Such as there being the anti-herbicide genes such as picture glyphosate (glyphosate) or glufosinates (phosphinothricin), as the antibiotic resistance gene of kantlex (Kanamycin), G418, bleomycin (Bleomycin), Totomycin (hygromycin), paraxin (chloramphenicol) etc., but be not limited to this.
In recombinant vectors of the present invention, promotor can be CaMV35S, Actin muscle, ubiquitin, pEMU, MAS or histone promotor, but is not limited to this.Term " promotor " refers to the DNA upstream region of structural gene, refers to the DNA molecular for the combination of transcriptional start RNA polymerase." plant promoter " is can be in the promotor of vegetable cell transcriptional start." constitutive character (constitutive) promotor " is the activated promotor of tool under most of envrionment conditions and growth conditions or cytodifferentiation.Because the selection of transformant can be formed by various tissues under the various stages, so constitutive character promotor is can be preferred in the present invention.Therefore, constitutive character promotor does not limit the possibility of selection.More preferably, can be ubiquitin promoter, but be not limited to this.
In recombinant vectors of the present invention, can use common terminator, such as the terminator etc. that has octopine (Octopine) gene of nopaline synthase (NOS), paddy rice α-amylase, RAmy1A terminator, phaseoline (phaseoline) terminator, Agrobacterium tumefaciens (Agrobacterium tumefaciens), but be not limited to this.About the necessity of terminator, be conventionally considered to can improve determinacy and the efficiency of vegetable cell transcription in these fields.Therefore the terminator, using in content of the present invention is very preferred.
In addition, the invention provides the plant materials that transforms expression of plants recombinant vectors of the present invention.The host cell that carrier of the present invention can be stabilized in to prokaryotic cell prokaryocyte and cloning and expression continuously can be used any host cell being disclosed in the sector, for example, bacterium and bacterial strain etc. in the intestines such as the Bacillus strain such as e. coli jm109, e. coli bl21, intestinal bacteria RR1, intestinal bacteria LE392, intestinal bacteria B, intestinal bacteria X1776, intestinal bacteria W3110, Bacillus subtilus, bacillus thuringiensis and Salmonella typhimurium, serratia marcescens and multiple pseudomonas kind.In addition, carrier of the present invention is transformed in eukaryotic situation, as host cell, can use yeast saccharomyces cerevisiae (Saccharomyce cerevisiae), insect cell, human body cell (for example, Chinese hamster ovary celI strain (Chinese hamster ovary cell (Chinese hamster ovary)), W138, BHK, COS-7,293, HepG2,3T3, RIN and mdck cell strain) and vegetable cell etc.Host cell is preferably vegetable cell, and more preferably paddy rice (Oryza sativa), most preferably is Eastern Jin Dynasty paddy rice, but is not limited to this.
For of the present invention, carrier is transported to for the method in host cell, if in the situation that host cell is prokaryotic cell prokaryocyte, can uses CaCl
2method, Hanahan method and electroporation method etc. are implemented.In addition, host cell is in eukaryotic situation, can use the methods such as the treatment process of microinjection, calcium phosphate precipitation method, electroporation method, liposome-mediated conversion method, diethylaminoethyl dextran and particle gun that carrier is injected in host cell.
In addition, the invention provides the method that changes the profile (architectures) of plant by level in adjusting OsMPT polypeptide cell.
OsMPT polypeptide of the present invention is at pulvinus (pulvinus) tissue specific expression of plant, is that the adjusting of the angle that is attached to stem to adjusting or the leaf of tillering angle is relevant, and above-mentioned OsMPT function by people of the present invention by open for the first time.OsMPT is preferably the polypeptide of the aminoacid sequence with SEQ IDNo.1.On the other hand, aforementioned polypeptides can be for the function equivalent with the polypeptide of the aminoacid sequence shown in SEQID No.1.For " function equivalent ", just as mentioning in above-mentioned, refer to the polypeptide in fact with OsMPT of the present invention with identical activity.At this, " having in fact identical activity " refers to plant structure and determines relevant activity.Above-mentioned functions coordinator, for example, comprise that the part in the amino acid of the aminoacid sequence shown in SEQ ID No.1 is substituted, disappearance or additional variant amino acid sequence body.Aminoacid replacement more preferably conservative property replaces.The conservative example replacing of amino acid existing in nature is as follows: aliphatic amino acid (glycine, L-Ala, proline(Pro)), hydrophobic amino acid (Isoleucine, leucine, α-amino-isovaleric acid), die aromatischen Aminosaeuren (phenylalanine, tyrosine, tryptophane), acidic amino acid (aspartic acid, L-glutamic acid), basic aminoacids (Histidine, Methionin, arginine, glutamine, l-asparagine) and sulfur-containing amino acid (halfcystine, methionine(Met)).Amino acid whose disappearance is more preferably positioned at the position that there is no direct relation with the activity of OsMPT of the present invention.
In addition, in the scope of above-mentioned functions coordinator, also comprise the basic framework of OsMPT and keep its physiologically active and polypeptide redundant organism that part chemical structure is made a variation.For example, in order to change the stability of polypeptide in the present invention, storage property, the structure modify of volatility or solubleness etc. and maintain physiologically active, and the fused protein forming after merging with other oroteins etc. is also contained in this.
In the present invention, the profile of plant refers to external structure or the form of plant, the tissue or the form separately that organ has and the whole form being combined to form thereof that represent the plants such as leaf, stem, root, flower, more preferably, the angle or the leaf that represent the connection section of tillering angle blade and leaf sheath are attached to the angle on stem, but are not limited to this.
In the present invention according to level in the cell of SEQ ID No.1 and function coordinator thereof, the part or whole length and the amplitude that are mainly above-mentioned tissue and organ can change, therefore, in the present invention, the variation main manifestations of plant profile is according to the part of pulvinus (pulvinus) tissue or whole variation.
In above-mentioned cell, level is the amount that phalangeal cell exists, and those skilled in the art can go to regulate its amount by multiple disclosed method.For example, in cell level can by transcription stage or the adjusting of transcribing in latter stage regulate, but be not limited to this.Adjusting in transcription stage can be implemented by the following method.The method of the expression for intensifying genes well known by persons skilled in the art, for example, prepare in promotor SEQ ID No.1 or connect the recombinant expression vector of the gene that SEQ ID No.1 function coordinator is encoded, thereby strengthen the method or at SEQ ID No.1 or gene that the function coordinator of SEQ ID No.1 is encoded around of the expression of said gene, insertion can be strengthened the method for the expression adjusting sequence of said gene, or the method for inhibition of gene expression, for example evoked promoter or gene locus sudden change, hinder the method for the function of promoter activity or protein, the method of antisence gene (antisense), RNAi or microRNA (microRNA) method etc.
The adjusting of transcribing in latter stage can be implemented by the following method.Well known by persons skilled in the art for strengthening or the method for the expression of arrestin matter, for example, strengthen or suppress to using the method for the stability of the mRNA that the gene of the function coordinator of coding SEQ ID No.1 or SEQ ID No.1 transcribes as model, strengthen or the method for stability or the active method of reinforcement or arrestin matter or polypeptide of arrestin matter or polypeptide.
More concrete example as aforesaid method, can by conversion encode as group 1 include subtype (group 1 intron type), M1RNA type, tup (hammerhead) type or hair clip (hairpin) type or with the DNA sequence dna of the microRNA type of the mRNA effect of transcribing, or conversion contains with the DNA of the same or similar sequence of target-gene sequence and induces co-suppression (cosuppression).
Preferably, can by increase or reduce the method for expression of the polynucleotide of coded polypeptide regulate SEQ ID No.1 in the present invention polypeptide or with its cell that is coordinator in level, preferably increase the expression of above-mentioned polynucleotide.The method of these increases or minimizing can be used respectively method known to those skilled in the art, for example, can be by connecting the recombinant expression vector of the polypeptide of SEQ ID No.1 or the polynucleotide of its coordinator of encoding in preparation promotor, thereby strengthen its expression, or can pass through to connect the recombinant expression vector for the antisense polynucleotides of above-mentioned polynucleotide in preparation promotor, thereby reduce its expression.Above-mentioned polynucleotide preferably consist of the base sequence shown in SEQ ID No.2.
Above-mentioned plant can be the angiosperms such as paddy rice, wheat, barley, oat, rye, turf, bamboo shoots, corn, sugarcane, maize, taro, asparagus, onion, garlic, green onion, leek, mountain garlic, Chinese yam, ginger, soybean or rape but be not limited to this.Be preferably paddy rice, more preferably Eastern Jin Dynasty paddy rice.The tissue that is subject to OsMPT protein expression variable effect in above-mentioned plant can be pulvinus.
In addition, the invention provides the preparation method of the plant that profile is changed and plant that the profile prepared by the method changes.Aforesaid method comprises and uses OsMPT or the step to the polynucleotide conversion of plant of encoding with its function coordinator.Preferably, above-mentioned polynucleotide can consist of the base sequence shown in SEQ ID No.2.The conversion of plant can be implemented by transformation technology well known by persons skilled in the art.Preferably utilize the method for transformation of edaphic bacillus, via Particle Bombardment Transformation method (microprojectile bombardment), electroporation (electroporation), PEG mediates fusion method (PEG-mediated fusion), microinjection (microinjection), liposome-mediated conversion method (liposome-mediated method), conversion method (In planta transformation) in plant materials, vacuum infiltration method (Vacuum infiltration method), floral meristem pickling process (floral meristem dipping method), Agrobacterium spraying method (Agrobacteria spraying method), preferably utilize the method for transformation of edaphic bacillus, most preferably utilize edaphic bacillus strain ' the method that transforms of LBA4404', but be not limited to this.
Now, above-mentioned polynucleotide can be for being operatively coupled on the state in promotor, so that can express in conversion of plant, for example, can be the form of the recombinant plant expression vector that is operably connected with promotor.' (operably linked) is operably connected ' refers to that a nucleic acid fragment is combined with other nucleic acid fragment, and its function or expression are subject to the impact of other nucleic acid fragment, and above-mentioned promotor and recombinant expression vector are as described above.
The vegetable cell transforming can be liquid culture, callus, protoplast culture thing, can be also tissue or the plant that is divided into plant.The cultivation of vegetable cell is after sterile culture, to cultivate by a part for plant is carried out under suitable condition from parent separation, can the liquid culture of using-system fragment, any modes well known by persons skilled in the art such as the callus culture of tissue fragment, protoplast cultures, its culture condition and method can be used condition well known by persons skilled in the art and method to implement.By the described plant cell differentiation of having cultivated, be that plant refers to the vegetable cell of having cultivated of callus, protoplastis form is induced to differentiation under suitable condition, thereby be divided into tissue or the plant of plant, its differentiation condition and method can be implemented by condition well known by persons skilled in the art and method.
Above-mentioned plant can be the angiosperms such as paddy rice, wheat, barley, oat, rye, turf, bamboo shoots, corn, sugarcane, maize, taro, asparagus, onion, garlic, green onion, leek, mountain garlic, Chinese yam, ginger, soybean or rape, but is not limited to this.Be preferably paddy rice, more preferably Eastern Jin Dynasty paddy rice.
In addition, the invention provides a kind of OsMPT of comprising gene for changing the composition of plant profile.Described OsMPT gene specifically expressing in pulvinus, thereby be attached in the Angular correlation composition of the present invention on stem with regulating angle or the adjusting leaf of tillering angle, adjusting leaf segment, above-mentioned OsMPT gene preferably, can consist of the base sequence of SEQ ID No.2 or SEQ ID No.3.In composition of the present invention, above-mentioned OsMPT gene can be included in the gene that in OsMPT gene order, specific base sequence is inserted into, replaces, lacks.
In addition, the invention provides the preparation method of the plant of output increase, it comprises the step of utilizing OsMPT gene-transformed plant.
Above-mentioned term " output " refers to the measurable turnout with economic worth, typically relevant to specific crop area and period.Each plant materials part according to directly with size and or weight assess output, or actual output is the annual production of every acre of crop, this acre deciding divided by plantation that be gross production (comprising the turnout of having gathered in the crops and be evaluated)." output " of plant can with the nutritive issue biomass (root and/or young stem biomass) of plant materials, reproductive organ, and/or person propagulum (for example, seed) is relevant.
For example, the output of paddy rice increase by as lower one with on represent: the quantity of per hectare or acre plant materials increases; The increase of every plant materials fringe grain (fruit) number; The increase of the granule number in spike number, every fringe, the weight of every particle, thousand corn weight, fruit length/diameter; The increase of seed-setting rate (full seed number/whole seed number x100).
Above-mentioned term " increase " can exchange and use with " raising " or " reinforcement ", the check plot plant that refers to and define in the present invention is compared and at least improves 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, be preferably at least 15% or 20%, preferred output and/or the growth of saying more than 25%, 30%, 35% or 40%.
In addition, the invention provides the plant of the output increase of preparing by aforesaid method.
Above-mentioned plant can be paddy rice, wheat, barley, oat, rye, turf, bamboo shoots, corn, sugarcane, maize, taro, asparagus, onion, garlic, green onion, leek, mountain garlic, Chinese yam, ginger, soybean, or the angiosperms such as rape, but be not limited to this.Be preferably paddy rice, more preferably Eastern Jin Dynasty paddy rice.
In addition, the invention provides the composition for increasing plant biomass that comprises OsMPT gene.In composition of the present invention, above-mentioned OsMPT gene preferably consists of the base sequence of SEQ ID No.2 or SEQ ID No.3.In composition of the present invention, above-mentioned OsMPT gene can comprise the gene that in OsMPT gene order, specific base sequence is inserted into, replaces, lacks.
Below, by embodiment, illustrate in greater detail the present invention.These embodiment are only for, illustration the present invention, and therefore content of the present invention is not subject to the restriction of following embodiment.
Experimental technique
1. vegetable material and growth conditions
Eastern Jin Dynasty paddy rice is as wild-type check plot.Ds transposon gene mutant library (pool) is for the separation of OsMPT gene mutation body.In the BR mutant using in the present invention, d61-1 mutant is available from loose ridge (Matsuoka) professor of Japan Nagoya university, and d11-1 and d2-2 mutant are available from the Gohuijong professor of Seoul National University.Plant is in the rice field of Korea S, to cultivate from June to October, at 26 ℃ of temperature of growth case (growth chamber), under/8 hours dark photoperiods of illumination in 16 hours, cultivates.
2. mutant and recovery mutant are separated
The specific gene of paddy rice pulvinus (pulvinus) is identified by OsMPT gene Ds insertosome.The sequential analysis that Ds inserts the flank region at position shows in the 3rd C2H2 Zinc finger domain, to be inserted into transposon (Fig. 2) in second exon of OsMPT.Used the RT-PCR of gene-specific primer to show that transcript is not expressed (Fig. 2) in gene mutation body.In order to obtain other gene mutation body allelotrope and to recover mutant allele, the OsMPT(OsMPT:Ds that comprises Ac) by tissue culture, be used for atomization more from generation to generation.11 new transposed Ds of OsMPT gene locus insert gene mutation body, recover mutant separated from aftergrowth (Fig. 2).
3.OsMPT cDNA cloning
Total length OsMPT cDNA is separated by cDNA end rapid amplifying polymerase chain reaction (R ace PCR) and reverse transcriptional PCR (RT-PCR).Comprise UTR(non-translational region) 5' and 3' end by being cloned of Race PCR, increase by RT-PCR in other position.The primer pair using is as follows: to 5'Race-PCR, be 5'-acctgtccgactccagcagcgtcctcg-3'(SEQ ID No.4); To 3'Race-PCR, be 5'-gtacaacttgtacgttcacgtgtgta-3'(SEQ ID No.5); To RT-PCR, be 5'-cggcatcaagaagcacttc-3'(SEQ ID No.6) and 5'-ggatgatggtgatgatgccgca-3'(SEQ ID No.7).Race-PCR cDNA is used BD SMART
tMrACE cDNA Amplifica tion Kit(Clontech, the U.S.) and synthetic.RT-PCR cDNA is that the RNA by SAM perienchyma is used SuperscriptII reversed transcriptive enzyme (hero company, the U.S.) and preparation.
4. cross the production of expressing transformant
In order to produce OsMPT, cross expression transformant, constitutional formula ubiquitin (Ubiquitin) promotor is merged by cloning and OsMPT full-length cDNA by binary vector pGA1611.Above-mentioned structure transforms edaphic bacillus (Agrobacterium) bacterial strain ' LBA4404 ', and imports to the callus introducing of Eastern Jin Dynasty paddy rice, generates transformant.
5.RNA is quantitative
Total cell RNA is to use TRIZOL reagent (TRIZOL reagent) (MRC, http://www.mrcgene.com/) or plant RNA extraction test kit (RNeasy Plant Mini Kits) (Qiagen, http://www.qiagen.com/) separated.For quantitative RT-PCR, article one, chain cDNA has been used Moroni murine leukemia virus (MMLV, Moloney murine leukemia virus) reversed transcriptive enzyme ribonuclease H (RNaseH) (Japanese Japan weaving, http://www.toyobo.co.jp/e/) according to the guide of manufacturers, synthetic in 25 μ L reaction mixtures of the RNA that comprises deoxyribonuclease (DNase) processing and Oligo dT 12-18 primer (hero company, http://www.invitr-ogen.com/).Above-mentioned reactant (1 μ L) is for pcr amplification.ACT1 mRNA(Actin muscle 1) with the reference material that acts on quantitative cDNA.The primer pair using is as follows: to ACT1, be that [McElroy etc. 1990, molecular biology of plants 14:163-171(McElroy etc. 1990, Plant Mol.Biol.14:163-171)] 5'-CGAGGCGCAGTCCAAGAG-3'(SEQ ID No.8) and 5'-CCCAGTTGCTGACGATACCA-3'(SEQ ID No.9); To OsMPT, be 5'-CGGCATCAAGAAGCACTTC-3'(SEQ ID No.10) and 5'-ggatgatggtgatgatgccgca-3'(SEQ ID No.11).Reaction is the initial stage modification of carrying out at 95 ℃ 5 minutes, then at 94 ℃, reacts 15 seconds, reacts 30 seconds, and at 72 ℃, react 40 seconds at 60 ℃.The cycle index of using in each amplification is as follows: ACT1,25 circulations; OsMPT, 32 or 35 circulations.The PCR product of amplification is separated in 1.5% sepharose.
6. phenotype analysis
The analysis of 6-1. phenotype is prepared
Investigate OsMPT and crossed the expression body performance in rice field in the wild.Selecting two conversion systems (OX14 and OX19) breeds.For on-the-spot test, by two independently two systems separately of transformant (OX14 and OX19) and non-transformed contradistinction systems (WT(OX14) and WT(OX19)) plant side by side.After conversion, in the next generation, can be separated into T-DNA and without the system of T-DNA, the system representation that has T-DNA is OX14 or OX19, be WT(OX14 without the system representation of T-DNA) or WT(OX19).Two kinds of different density (30 * 15cm(23.3 plant/m that plant for 6 systems altogether
2) and 15 * 15cm(46.7 plant/m
2) plant experiment.Body cultivates plants in the GMO field experiment of close sun (Korea S's place name) Village Revitalization Office.According to the paddy rice standard cultivating method of Village Revitalization Office, cultivate and manage.The size of each trial plot (plot) is 2 * 1.5m, according to random district group (randomized blocks), carries out 2 experiments repeatedly.In each trial plot, 70 and 140 plant individuals are planted according to standard density and high-density altogether.Except edge plant materials, 3 individualities that close on from each trial plot results.According to standard, plant the non-transformed contradistinction system of OX19 (WT(OX19) of distance plantation) because lodging (lodging) disappears, thus be excluded.
6-2. observes the growth form that OsMPT crosses expression system in rice field
In the wild under group's state in rice field in order to verify that OsMPT crosses growth form and the phenotypic expression of expression system, in GMO rice field, at two kinds, plant under density, in standard, plant apart from (30 * 15cm) and dense planting and plant the laggard row observation of the lower plantation of distance (15 * 15cm).
6-3. parametric measurement
From standard density rice field and high-density rice field, gathered in adjacent column the plant materials of approximately 30 individual and 60 individualities.But with standard, plant the non-transformed contradistinction system (WT(OX19) of the OX19 of distance plantation), in above-mentioned two rice fields because lodging (lodging) disappears.Measured following harvesting correlative factor: cane length (culm length), spike length (panicle length), plant materials panicle quantity (number of panicles per plant), small ear sum (total number of spikelets), fertile seed sum (total number of fertile seeds), particle gross weight (total grain weight).
Embodiment 1.OsMPT encodes to having the protein of zinc fingers
OsMPT encodes to having the nucleoprotein of 4 zinc fingerses (C2H2C2H2C2HC C2HC).This motif is similar to the zinc fingers that the corn florescence regulatory gene delivered " Id1(Indeterminate1) " has (Colasanti etc. 2006, BMC genomics 7,158(Colasanti etc. 2006, BMC Genomics7,158)).
Embodiment 2.OsMPT is the function autoploid (orthologue) that gravity reacts relevant Arabidopis thaliana (Arabidopsis) SGR5
According to reports, Arabidopis thaliana SGR5(shooting gravity reaction 5(Shoot Gravity Response5)) with gravity react relevant (Morita etc. 2006, plant magazine 47,619-628(Morita etc. 2006, Plant J.47,619-628)).OsMPT has 42% consistence and 54% similarity (Fig. 1) with SGR5 in amino acid levels.At present, for the relevant following data of gravity reaction of report OsMPT are prepared: the metaxylem of OsMPT, the connection section of pulvinus (gravity sensing organ gramineous) and laminar joints(blade and leaf sheath) in expression specifically.The geotropism of gene mutation body extremely reduces.The mistake expression body of OsMPT shows the geotropism reaction of raising.
Embodiment 3.OsMPT gene mutation body and cross expression body to tillering and laminar joint angle plays reverse effect
OsMPT regulates tillers and the angle of the connection section of blade and leaf sheath.Gene mutation body tiller and connection section (Fig. 3) angle of blade and leaf sheath broadens.On the contrary, too much produce a tillering angle that cognition demonstration is narrow of OsMPT and be grown to upright leaf (Fig. 4).OsMPT crosses phenotype and the BR(brassinosteroid of expression body) gene mutation body similar (Fig. 5).Genetic analysis represents, OsMPT gene mutation body with respect to BR signal gene mutant d61-1 and BR dcc gene mutant d11-1, is positioned at bottom (epistatic) (Fig. 6) when determining tillering angle.OsMPT demonstrates in the bottom of BR effect and works.
The agriculture practicality of embodiment 4.OsMPT
Rice yield is subject to the very big impact by tiller number, tillering angle, internode elongation, panicle form and leaf angle (leaf angle) defined plant profile (architectures).Therefore in above-mentioned factor, tiller and leaf angle can determine that leaf surfaces sunlight incident efficiency is important factor.Up to the present, concerning paddy rice, BR defect is the unique known facts that causes erect type plant.Verified while going out to utilize BR-dcc gene mutant dense planting erect type rice plant paddy rice, have higher output (Sakamoto etc. 2006, Nature Biotechnol 24,105-109(Sakamoto etc. 2006, Nat.Biotechnol.24,105-109)).OsMPT works in the bottom of BR effect, therefore in OsMPT transformant, does not expect the change of BR homeostasis (homeostasis).In addition, the synthetic or Inhibitory signal gene than BR, OsMPT-crosses expression strategy and is more prone to.
Embodiment 5. OsMPT in rice field crosses the phenotype analysis of expression system
In order to verify growth and phenotype expression in group, in GMO rice field, to two kinds, plant density and cultivate observation.As shown in Figure 8, OsMPT crosses expression system and in standard, plants distance (30x15cm) and dense planting and show obvious vertical growth in planting apart from (15x15cm).OsMPT system is planted in distance because the space between plant is secure in dense planting, so line space is also very obvious.In the situation of planting culture, there is lodging phenomenon in general paddy rice (Eastern Jin Dynasty paddy rice) when through rainy season, but OsMPT demonstrates resistivity to lodging, is therefore considered to that planting culture is had to fabulous adaptive faculty.
The phenotype evaluation result of conversion of plant is represented to be listed in table 1 according to the mean value of each index and standard deviation.The mean value of the plant materials number of enumerating in each row that value is table 1.
Table 1
Harvest index 2010
PH, plant height; PN, spike number; TNS, seed sum; NFG, solid seed sum; TGW, total grain is heavy; AVE, mean standard deviation; %, wild-type compatriot's (sibling) MPT1 ratio.
Spike length (panicle length) OX19 system in OsMPT system grows approximately 5 ~ 20%.Compare with non-transformed contradistinction system, OsMPT ties up to seed sum in high-density rice field than in standard density rice field have been increased.Seed sum is compared with non-transformed contradistinction system, height approximately 6 ~ 30% in OsMPTOX system.The setting percentage of the plant seed in the OsMPT plant comparison with standard density rice field in high-density rice field is high.OsMPT compares with non-transformed contradistinction system and has produced nearly 6 ~ 20% solid seed.The grain heavy (grain weight) of every plant materials is increased to 5% in OsMPT system.
OsMPT OX ties up to high-density and has shown good performance in planting.The spike length of OsMPT OX (panicle length), grain number per spike (grain number), solid seed (filled seeds) and total grain heavy (grain weight) have increased.
Claims (8)
1. in the polypeptide cell consisting of the aminoacid sequence shown in SEQ ID No.1 adjusting, level regulates the tillering angle of paddy rice or the method that leaf is attached to the angle on stem.
2. method according to claim 1, is characterized in that, in described cell, the adjusting of level is to implement by increasing or reduce the expression of the polynucleotide of coding said polypeptide.
3. method according to claim 2, is characterized in that, described polynucleotide consist of the base sequence shown in SEQ ID No.2.
4. method according to claim 1, is characterized in that, described cell is pulvinus histocyte.
5. regulate tillering angle or regulate leaf to be attached to a preparation method for the paddy rice of the angle on stem, it comprises the step of the gene-transformed plant of the OsMPT protein that derives from paddy rice (Oryza sativa) that the aminoacid sequence of utilization coding SEQ ID No.1 forms.
6. the composition of the gene of the OsMPT protein that derives from paddy rice (Oryza sativa) of an aminoacid sequence formation that comprises coding SEQ ID No.1 is regulating tillering angle or the leaf of paddy rice to be attached to the application in the angle on stem.
7. the preparation method of the paddy rice that an output increases, it comprises the gene-transformed plant of the OsMPT protein that derives from paddy rice (Oryza sativa) of the aminoacid sequence formation of utilizing coding SEQ ID No.1, and the step that the plant of conversion is planted with high-density.
8. the application of the composition of the gene of the OsMPT protein that derives from paddy rice (Oryza sativa) of an aminoacid sequence formation that comprises coding SEQ ID No.1 in increasing rice yield.
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PCT/KR2010/009447 WO2011090272A2 (en) | 2010-01-22 | 2010-12-28 | OsMPT GENE MODIFYING PLANT ARCHITECTURE (PLANT SHAPE) AND INCREASING YIELD, AND USE THEREOF |
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CN104878020B (en) * | 2015-04-29 | 2017-09-15 | 华中农业大学 | A kind of gene for controlling the upright sexual development of rice leaf and its application |
CN108017697B (en) * | 2017-12-25 | 2020-06-30 | 中国科学院遗传与发育生物学研究所 | Plant tillering angle related protein HSFA2D, and coding gene and application thereof |
CN109082437B (en) * | 2018-07-03 | 2020-11-13 | 浙江大学 | Method for increasing tillering quantity of barley |
CN115851755B (en) * | 2022-07-11 | 2024-05-24 | 内蒙古农业大学 | Mongolian hybrid agro-grass tillering angle regulating factor and application thereof |
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US9150875B2 (en) | 2015-10-06 |
US20120284875A1 (en) | 2012-11-08 |
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